Assessment of feedlots cattle in the development and spread of Vancomycin resistant Enterococci

(1)

Assessment of feedlots cattle in the

development and spread of Vancomycin

resistant Enterococci

Frank Eric Tatsing Foka

Orcid ID:

0000-0001-6577-2827

Thesis submitted in fulfilment of requirements for the degree of

Doctor of Philosophy in Biology

at the North-West University

Promoter: Professor C.N. Ateba

Examination: December 2019

Student number: 28035348

(2)

SUMMARY

Enterococci are commensals of the gastrointestinal tract of warm-blooded animals. They have been incriminated in a wide range of community-aquired infections and nosocomial diseases, especially in immunocompromised individuals and stressed animals. If enterococci have been able to survive and thrive in different ecological niches to date, it is due mainly to their outstanding capability to adapt to these various environmental settings by incorporating into their genetic constitution, resistance genes to many currently used antimicrobials. The widespread use of antibiotics in industrial animal husbandry has contributed significantly to the emergence of resistant isolates, such as vancomycin-resistant enterococci (VREs) worldwide and in South Africa, specifically. Despite the fact that Avoparcin, which is a glycopeptide and analogue of vancomycin, was identified as the source of the emergence of VREs and was forbidden worldwide in industrial animal farming decades ago, VREs are regularly screened in environmental samples in the North West Province, South Africa. This study, therefore, sheds some light on the various reasons why VREs are continuously detected in environmental samples in the North West Province of South Africa. Very few studies in South Africa have focused on the involvement of cattle feedlots in the spread and dissemination of such strains in the environment. In this regard, we aseptically collected 384 faecal samples, 24 drinking troughs water and 24 soil/liter samples from six registered feedlots of the North West Province, South Africa. Thereafter, we used biochemical and molecular methods to identify and categorise the isolated enterococci. Furthermore, we determined their antibiotic resistance and their virulence profiles with phenotypic and genotypic methods as well as Next Generation Sequencing platforms. Five humdred and twenty-seven (527) presumptive isolates were recovered, while two hundred and eighty-nine (289) isolates were confirmed as members of the genus Enterococcus. Species specific PCR protocols were used to identify them as E. faecalis (9%), E. faecium (10%), E. durans (69%), E. gallinarum (6%), E. casseliflavus (2%),

(3)

E. mundtii (2%) and E. avium (2%). Vancomycin resistance genes were detected through PCR

assays in 176 isolates, precisely vanA (62%), vanB (17%) and vanC (21%). Moreover, four Tetracycline efflux pump genetic determinants were also detected through PCR methods in 138 of the screened VREs and these included tetK (26), tetL (57), msrA/B (111) and mefA (9). All the VREs of this study were multidrug resistant and four antibiotic resistance profiles were identified. Furthermore, cylA, hyl, esp, gelE and asa1 virulence genes were detected in 86 VREs, with some harbouring more than one virulence gene. The cluster analysis of the VREs of this study was based on the diameters of the antibiotic inhibition zones and revealed a similar exposure history to the antibiotics tested. Data on antimicrobials currently used in the feedlots under investigation, either for prophylactic purposes or for therapeutic purposes as well as for growth promoting purposes, was collected. Data was assessed along with the bioinformatical analysis of the whole genome sequences of VR E. durans strain NWUTAL1 and VR E. gallinarum strain S52016, isolated from feedlot cattle faeces and feedlot soil/liter samples

respectively. The data derived from these analyses revealed that some of the antibiotics used in the feedlots, were responsible for the resurgence of VREs. Specifically, plasmids with resistance genes to Tetracycline, Tylosin and Erythromycin were detected in these VREs. These antibiotics wield a sort of selective pressure on their specific antibiotic resistance genetic determinants that are co-selected with Vancomycin resistance genes. The fact that potentially pathogenic multidrug resistant VREs were detected in this study, demonstrates that practices such as the extensive usage of antibiotics in industrial animal husbandry, have a significant impact on the environment and its ecological niches. This may, consequently, affect humans and other living organisms since it enhances the availability of antimicrobial resistance genes in the environment, which is taken up and exchanged among commensals that were not initially harmful. Since such commensals find their way through direct and indirect contacts into the

(4)

food chain, issues of this nature cannot be undermined, especially in the context of South Africa, where the occurrence of AIDS/HIV and diabetes is high.

(5)

DECLARATION

I, the undersigned, declare that the thesis hereby submitted to the North-West University, Mafikeng Campus, for the degree of Doctor of Philisophy (PhD) in Biology and the work contained herein, is my own original work in design and execution. I further declare that the thesis has not previously, in its entirety or in part, been submitted to any other institution for an academic qualification. All materials used in the study have been duly acknowledged.

Signed at ... on this ... Day of ... 2019

________________

F. E. Tatsing Foka

(Student)

Signed at ... on this... Day of ... 2019

_______________

Professor C.N. Ateba

(6)

DEDICATION

I dedicate this study to my parents, Mr and Mrs Foka, who, against all odds, gave me the headstart I needed in life by providing and teaching me the importance of education. Your unconditional love, care and support throughout every single step of my life, made me stronger as I faced every challenge. Without you, I would not have realised this endeavour.

(7)

ACKNOWLEDGEMENTS

I wish to thank the Heavenly Father, for my life and most especially, my studies; you stood firm and next to me and never failed me. You are worthy my Lord to receive Glory for you are above all living beings and your power is beyond any other power. All things shall pass but you were, you are and you will forever be.

I would like to express my gratitude to my supervisor, Professor C.N. Ateba, for giving me the opportunity to do a PhD in his research group, for his constant support, patience and encouragement throughout my studies. It has truly been inspiring and uplifting working with him throughout these years.

Institutionally, I would like to acknowledge the financial support provided by the North-West University Institutional Bursary and the North-West University merit bursary. Moreover, I acknowledge all the members of staff of the School of Biological Sciences, North-West University, Mafikeng Campus. I appreciate their support in the realisation of this project. The assistance offered by Dr Ayanbgero Ayansina, Mr Peter Montso, Mr Alayande Kazeem, Mr Akinola Stephen, Mr Christ Donald Kaptchouang and my colleagues of the Molecular Microbiology laboratory is fully appreciated. I would also like to acknowledge Professor Noutchie Okouomi Suarez Clovis and Dr Yah S. Clarence, for their kind assistance and support during this journey. I am eternally grateful to Professor Carlos Bezendehout, Dr Charlotte Minnie and Mr Rudolph of the North-West University, Potchefstroom Campus, for the guidance and support provided in the 16S rRNA and NGS analysis of my isolates.

I also deeply appreciate Dr K. S. Bet, Department of Geography, North-West University, for providing a map of my sampling points and the Cameroonian community in Mafikeng, for their constant words of encouragement.

Finally, I thank my parents and my sisters, for their prayers, love and unending support throughout this journey. I would also like to express my appreciation to Mr and Mrs Chongang,

(8)

for their support and prayers throughout these years. To my wife and my children, I know how hard it must have been for you to bear my absence throughout these years, without your love and your support, I would not have made it.

(9)

LIST OF TABLES

Table 2.1: Species of the genus Enterococcus………29

Table 2. 2: Antibiotics used as growth promoters in the European community, past and present ... 64

Table 2. 3: Growth promoters used in intensive animal rearing in South Africa ... 65

Table 3. 1: Antimicrobials used in South Africa as growth promoters ... 108

Table 3. 2 : Antibiotics sold in the private and public sector (from 2014 to 2016) ... 112

Table 3. 3: Actions prioritised at the Third World Healthcare Associated Infections Forum with respect to antimicrobial resistance. ... 115

Table 4. 1: Types of samples used in this study ... 131

Table 4. 2: Oligonucleotide primers used in this study ... 136

Table 4. 3: Distribution of enterococcal species per sampling site ... 139

Table 4. 4: Predominant multidrug resistance patterns observed among isolates ... 143

Table 4. 5: Virulence genes patterns in VRE isolates from different sampling sites ... 146

Table 4. 6: Cluster distribution of isolates ... 147

Table 5. 1: Assembly reports of E. durans NWUTAL1 and E. gallinarum S52016 genomes ... 165

Table 5. 2: Protein features of E. durans NWUTAL1 and E. gallinarum S52016... 172

(16)

LIST OF FIGURES

Figure 2. 1: Illustration of genetic elements involved in the spread of ARG as a result of

selective pressure from the use of antibiotics.. ... 54

Figure 2. 2: VanA resistance gene cluster and resistance mechanism. . ... 58

Figure 2. 3: Comparisons of several Vancomycin resistance clusters found in enterococci. . 62

Figure 2. 4: Transmission pathway of antimicrobial resitance genes ... 69

Figure 3. 1: Vancomycin resistance gene clusters and resistance mechanism. ... 103

Figure 3. 2: Transmission pathway of antimicrobial resistance within agriculture, the environment and the food-processing industry. ... 107

Figure 4. 1: Trend in Vancomycin resistance genes among enterococcal isolates from the feedlots ... 140

Figure 4. 2: Multiplex PCR positive isolates. ... 141

Figure 4. 3: Distribution of Tetracycline-resistant VREs... 142

Figure 4. 4: Tetracycline resistant VRE isolates... 142

Figure 4. 5: Proportions of antibiotic resistant VRE isolates ... 143

Figure 4. 6: Enterococcal strains with virulence genes ... 144

Figure 4. 7: Dendogram depicting the relationship between 72 multidrug resistant VREs isolated from the feedlots.. ... 148

Figure 5. 1: Subsystem analysis of strain NWUTAL1 and strain S52016 ... 173

Figure 5. 2: Circular graphical display of the distribution of the annotated genomes of strain NWUTAL1 and strain S52016 .. ... 175

(17)

Figure 5. 3: Phylogenetic tree determining the relationship between strains NWUTAL1, S52016 and other enterococci of the same species. ... 182

(18)

CHAPTER 1

(19)

Chapter one

General introduction and problem statement

1.1 Introduction

The beef industry is an important value-added enterprise worldwide and millions of farms and ranches benefit directly from the sale of slaughtered animals, thus contributing significantly to the economic output of their respective countries (Economou and Gousia, 2015). Enterococci are extremely versatile organisms that occur as commensals in the gastrointestinal tract of warm-blooded animals and humans (Aarestrup, 2000) and as opportunistic pathogens in immunocompromised persons (Acar et al., 2012). Also known to be ubiquitous organisms, enterococci can survive under adverse environmental conditions (Acar et al., 2012) and can, therefore, be isolated from environmental samples such as water, soil or plant surfaces (Ateba and Mohapi, 2013; Matlou et al., 2019). Their dominance in the digestive tract of warm-blooded animals and humans (Ateba and Maribeng, 2011), has resulted in some strains being used as potential indicators of faecal contamination in food and water (Ateba and Maribeng, 2011; Ateba and Mohapi, 2013).

The discovery of antibiotics was a corner stone in the evolution of humanity since antibiotics became life-saving substances for both animals and humans (Gonzalez-Zorn and Escudero, 2012). Chemotherapy had a positive and deep impact on the society since availability of antibiotics and antimicrobial agents prolonged life expectancy through the ability to treat common infections, thus allowing rapid population growth (Gonzales-Zorn and Escudero, 2012). Antibiotics have since then, become a key element not only in the success of surgical interventions, but also in successful intensive animal rearing (Acar and Moulin, 2012; Economou and Gousia, 2015). For instance, antimicrobials are extensively used in cattle rearing for prevention, control and treatment of infections; but most importantly, to improve

(20)

growth and feed efficiency (USFDA, 2014). Although it has been demonstrated somehow, that various types of resistance attributes and resistant bacterial strains were present long before the production and usage of antimicrobials (Acar et al., 2012), the extensive use of antimicrobial agents, especially antibiotics, has unfortunately, led to the rise of resistant isolates (Rzewuska et al., 2015). This has resulted in severe consequences on consumers and, therefore,

undermines the importance of the positive effects of antimicrobials in both animals and humans (Jui-Chang et al., 2005). Consequently, antibiotic-resistant bacterial strains have made therapeutic alternatives for the treatment of infections caused by multi-drug resistant organisms limited and their subsequent antimicrobial resistance genes (ARG’s) available in the environment (Tao et al., 2014). In fact, extended spectrum beta-lactamase-producing (ESBL) E. coli resistant to category I antibiotics, have been detected among farm animals in Canada

and the United States of America (Mollenkopf et al., 2012). In addition, Taucer-Kapteijin and his colleagues (2016) isolated Vancomycin resistant enterococci (VRE) from surface water intended for drinking water production in the Netherlands. Similarly, Vancomycin resistance genes have also been detected in VRE strains isolated from ground water intended for human consumption and food products in Mafikeng, South Africa (Ateba and Maribeng, 2011; Ateba and Mohapi, 2013).

Avoparcin, a growth promoter and analogous compound to Vancomycin (Bager et al., 1997), was banned worldwide due to its association with a high prevalence of VRE, both in faeces of exposed animals and in meat products (Bager et al., 1997; Wegener et al., 1998; Aarestrup, 2000). Despite this, the constant detection of VRE as well as the confirmation of Vancomycin resistance gene determinants in isolates from environmental samples, even in countries with strict drug usage policies and advance public health facilities, is a cause for concern (Borgen et al., 2002a; Borgen et al., 2002b). The detection of antimicrobial resistant bacteria has

(21)

controversy over antimicrobial resistant pathogens and the use of antimicrobials in food animals may also have severe trade implications on beef and food industries (Economou and Gousia, 2015). However, the detection of antibiotic resistance phenotypes and determinants does not always result from the usage of a particular antibiotic (Nilsson et al., 2009). For instance, Streptococcus pyogenes remains fully sensitive to Penicillin despite the presence of selective pressure (Bager et al., 1997) while E. coli is, most often, resistant to Chloramphenicol despite the removal of selective pressure (Ateba and Mohapi, 2013). In addition, due to co-selection of resistance determinants, problems associated with antibiotic resistance is worsened since resistance traits in bacteria cannot be reversed by discontinued use of a particular antibiotic (Gonzalez-Zorn and Escudero, 2012).

Based on the aforementioned, two types of Vancomycin resistance in enterococci have been demonstrated as follows: intrinsic; and acquired resistance (Aarestrup, 2000). Intrinsic resistance is characterised by low-level resistance to Vancomycin and this type of resistance is commonly detected in Enterococcus gallinarum, Enterococcus casseliflavus and Enterococcus flavescens (Jui-Chang et al., 2005). On the contrary, strains of E. faecium and E. faecalis and

less often, E. raffinosus, E. avium and E. durans, are known to display acquired resistance to Vancomycin (Jui-Chang et al., 2005), resulting from the acquisition of genetic determinants, either from other organisms or from the environment (Jui-Chang et al., 2005; Sónia et al., 2014).

1.2 Problem statement

Public awareness related to antimicrobial usage (AMU) in livestock continues to increase, as does continuing pressure for governments and industries to address these concerns, given the constant rise in antimicrobial resistance in bacteria (USFDA, 2014). In the past decades, Vancomycin was utilised to treat Methicilin Resistant Staphylococcus aureus (MRSA) worldwide while its analogue (Avoparcin) was used as a growth promoter in cattle feeds

(22)

(Aarestrup, 2000). Due to the emergence of VRE, resulting from the misuse of Avoparcin, the use of Vancomycin, as the first choice drug in the treatment of resistant enteroccocci infections, was affected while Avoparcin was banned (Bager et al., 1997; Wegener et al., 1998; Aarestrup, 2000; Nilsson et al., 2009). Despite the fact that Vancomycin and its analogue (Avoparcin) are currently not in use in both veterinary and human medicine worldwide and in South Africa, in particular, a number of scientific investigations revealed the presence of VRE as well as other resistant bacterial strains that possess VRGs from animals (Moneoang and Bezuidenhout, 2009; Bekele and Ashenafi, 2010), meat (Sudeep et al., 2014), ground and surface water (Ateba and Maribeng, 2011; Taucer-Kapteijin et al., 2016; Matlou et al., 2019) and fresh vegetables (Ateba and Mohapi, 2013). As a matter of fact, results obtained from two studies conducted in Mafikeng, North West Province, South Africa revealed that VRE strains from lettuce and spinach leaves collected from some supermarkets (Ateba and Mohapi, 2013) as well as those isolated from ground water, intended for human consumption (Ateba and Maribeng, 2011), were resistant to multiple antibiotics, including Amoxicillin, Ampicillin, Chloramphenicol, Teicoplanin, Tetracycline, Penicillin and Erythromycin. In addition, multidrug-resistant enterococci have also been detected in food-producing animals in South Africa (Moyane et al., 2013). Therapeutic difficulties presented by VRE, especially strains that portray high-level aminoglycoside resistance traits, outlines the need to determine the source of VRGs detected among enterococci. This study was, therefore, designed to determine the contribution of feedlots cattle in the development and dissemination of VRGs in the environment, thus, the importance of this study cannot be overemphasised.

1.3 Research questions

The following research questions were asked:

 Does antimicrobial usage in cattle feedlots promote the dissemination of Vancomycin resistance genes? and

(23)

 What are the causes of phenotypic and genotypic Vancomycin resistance in enterococci from feedlots environments?

1.4 Hypotheses

The following research hypotheses were stated in the study:

 H0: Antimicrobial usage in cattle feedlots has a strong impact on the availability and dissemination of Vancomycin resistance genes in the environment .

 H1: Vancomycin resistance genes are not expressed in VREs as a result of antimicrobial usage in intensive animal rearing.

1.5 Aim and objectives of the study

1.5.1 Aim of the study

The aim of this study was to assess the impact of antibiotic usage in cattle feedlots of the North West Province with regard to the development and spread of VRE strains in the environment.

1.5.2 Objectives of the study

The specific objectives of the study were to:

 Isolate Vancomycin resistant enterococci from faecal samples and drinking water troughs and soil samples of feedlots and feedlots cattle;

 Identify the species identity of VREs isolated through species-specific PCR protocols;

 Determine the genetic antibiotic resistance profiles of isolates through PCR protocols and disc diffusion assays;

 Determine virulence gene determinants of VRE isolates; and

 Assess genetic determinants involved in the propagation of Vancomycin resistance in VREs isolated through Whole Genome Sequencing.

(24)

REFERENCES

Aarestrup, F.M. (2000). Characterization of glycopeptide-resistant Enterococcus faecium (GRE) from broilers and pigs in Denmark: genetic evidence that persistence of GRE in pig herds is associated with coselection by resistance to macrolides. J. Clin. Microbiol. 38, 2774 – 2777.

Ateba, C.N. and Maribeng, M.D. (2011). Detection of Enterococcus species in groundwater from some rural communities in the Mmabatho area, South Africa: a risk analysis. Afr. J. Microbiol. Rev. 5 (23), 3930 – 3935.

Ateba, C.N. and Mohapi, M.I. (2013). Isolation of vancomycin resistant enterococci isolated from leafy vegetables (lettuce) from North West Province. Life Sci. 10 (4), 1163-1170. Acar, J.F. and Moulin, G. (2012). Antimicrobial resistance: a complex issue. Rev. Sci. Tech.

31, 23 – 31.

Acar, J.F., Moulin, G., Page, S.W. and Pastoret, P.P. (2012). Antimicrobial resistance in animal and public health: introduction and classification of antimicrobial agents. Rev. Sci. Tech. 31, 15 – 21.

Bager, F.M., Madsen, J.C. and Aarestrup, F.M. (1997). Avoparcin used as a growth promoter is associated with the occurrence of vancomycin-resistant Enterococcus faecium on Danish poultry and pig farms. Prev. Vet. Med. 31, 95 – 112.

Bekele, B. and Ashenafi, M. (2010). Distribution of drug resistance among enterococci and Salmonella from poultry and cattle in Ethiopia. Trop. Anim. Health Prod. 42, 857 – 864. Borgen, K., Sorum, M., Wasteson, Y., Kruse, H. and Oppegaard, H. (2002). Genetic linkage between erm(B) and vanA in Enterococcus hirae of poultry origin. Microb. Drug Resist. 8, 363 – 368.

Borgen, K., Wasteson, Y., Kruse, H. and Willems, R.J. (2002). Vancomycin-resistant Enterococcus faecium (VREF) from Norwegian poultry cluster with VREF from

(25)

poultry from the United Kingdom and The Netherlands in an amplified fragment length polymorphism genogroup. Appl. Environ. Microbiol. 68, 3133 – 3137.

Economou, V. and Gousia, P. (2015). Agriculture and food animals as a source of antimicrobial-resistant bacteria. Infect. Drug Resist. 8, 49 – 61.

Gonzalez-Zorn, B. and Escudero, J.A. (2012). Ecology of antimicrobial resistance: humans, animals, food and environment. Int. microbial. 15, 101 – 109.

Jui-Chang, T., Po-Ren, H., Hsiao-Mann, L., Hui-Jen, C., Shen-Wu, H. and Lee-Jene, T. (2005). Identification of Clinically Relevant Enterococcus Species by Direct Sequencing of groES and Spacer Region. J. Clin. Microbiol. 43, 235 – 241.

Matlou, D.P., Bissong M.E.A., Tchatchouang C.K., Adem, M.R., Foka, F.E.T. and Ateba, C.N. (2019). Virulence profiles of vancomycin-resistant enterococci isolated from surface and ground water utilized by humans in the North-West Province, South Africa: a public health perspective. Environ. Sci. Pollut. Res. 26 (15), 15105 – 15114.

Mollenkopf, D.F., Weeman, M.F., Daniels, J.B., Abley, M.J., Mathews, J.L. and Gebreyes, W.A. (2012). Variable within and between herd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle. Appl. Environ. Microbiol. 78, 4552 – 4560.

Moneoang, M.S. and Bezuidenhout, C.C. (2009). Characterisation of enterococci and Escherichia coli isolated from commercial and communal pigs from Mafikeng in the

North-West Province, South Africa. Afr. J. Microbiol. Res. 3 (3), 88 – 86.

Moyane, J.N., Jideani, A.I.O. and Aiyegoro, O.A. (2013). Antibiotic usage in food producing animals in South Africa and impact on human: antibiotic resistance. Afr. J. Microbiol. Res. 7 (24), 2990 – 2997.

(26)

Nilsson, O., Grekol, C., Top, J., Franklin, A. and Bengtsson, B. (2009). Spread without known selective pressure of a vancomycin-resistant clone of Enterococcus faecium among broilers. J. Antimicrob. Chemother. 10, 1 – 5.

Rzewuska, M., Stefanska, I., Kizerwetter-Swida, M., Chrobak-Cmiel, D., Szczygielska, P., Lesniak, M., and Binek, M. (2015). Characterization of extended-spectrum-beta-lactamases produced by Escherichia coli strains isolated from dogs in Poland. Pol. J. Microbiol. 64, 285 – 288.

Sónia, R., Ingrid, C., Nuno, S., Michel, H., Hugo, S., José-Luis C.M., Patrícia, P. and Gilberto, I. (2015). Effect of vancomycin on the proteome of the multiresistant Enterococcus faecium SU18 strain. J. Proteomics. 113, 378 – 387.

Sudeep, G., Bahadur, B.H., Lok-Raj, J. and Maheshwar, S. (2014). Prevalence of vancomycin-resistant enterococci species in minced buffalo meat of Chitwan, Nepal. Int. J. Appl. Sci. Biotechnol. 2 (4), 409 – 412.

Tao, C.W., Bing-Mu, H., Wen-Tsai, J., Tsui-Kang, H., Po-Min, K., Chun-Po, H., Shu-Min, S., Tzung-Yu, S., Tern-Jou, W. and Yu-Li, H. (2014). Evaluation of five antibiotic resistance genes in wastewater treatment systems of swine farms by real-time PCR. Sci. Total Environ. 496, 116 – 121.

Taucer-Kapteijin, M., Hoogenboezem, W., Heiliegers, L., Danny de Bolster, H. and Medema, G. (2016). Screening municipal wastewater effluent and surface water used for drinking water production for the presence of ampicillin and vancomycin resistant enterococci. Int. J. Hyg. Environ. Health. 16, 1 – 7.

US Food and Drug Administration Center for Veterinary Medicine. Judicious use of antimicrobials for beef cattle veterinarians. Available from: www.fda.gov/downloads/Animal veterinary/Safety health/Antimicrobial resistance/pdf. Accessed on June 10, 2016.

(27)

Wegener, H.C., Madsen, N. and Aarestrup, F.M. (1997). Isolation of vancomycin resistant Enterococcus faecium from food. Int. J. Food Microbiol. 35, 57 – 66.

(28)

CHAPTER 2

(29)

CHAPTER TWO

LITERATURE REVIEW

2.1 General characteristics of enterococci

Members of the genus Enterococcus were described for the first time in 1899 by Thiercelin as “coccoid-shaped bacteria from the human intestine”. The appellation “entérocoque” was then used to point out their intestinal origin, even though the term Streptococcus was still regularly used (Thiercelin, 1899). Later on classified as “group-D streptococci”, Sherman had a brilliant idea in 1937 as he developed a new method of classification of the genus Streptococcus into four main categories as follows: pyogenic; viridans; lactic and enterococci (Sherman, 1937; Salminen et al., 2004). Later on, in 1984, the term “Enterococcus” was introduced due to the difference demonstrated between Streptococci and Enterococci after DNA and DNA-RNA hybridisation experiments (Ogier and Serror, 2008). Based on the comparative analysis of the 16S rRNA gene sequences, 43 species of enterococci have been identified so far. The following enterococcal species are of medical importance:

Table 2. 1: Species of the genus Enterococcus

Group Examples of species in group

E. faecium group E. faecium, E. durans, E. hirae, E. mundtii, E. villorum, E. canis, E. azikeev;

E. faecalis group E. faecalis, E. haemoperoxidus, E. moraviensi, E. ratti;

E. avium group E. avium, E. malodoratus, E. pseudoavium, E. raffinosus, E. gilvus;

E. gallinarum group E. gallinarum, E. casseliflavus, E. flavescens;

(30)

E. saccharolyticus group E. saccharolyticus, E. sulfures;

E. cecorum group E. cecorum, E. columbae;

Source: Salminen et al., 2004

Enterococci are Gram-positive, catalase negative and facultative anaerobic cocci that occur either singly, in pair or in chains (Aarestrup, 2000). They are commensals of the intestinal microbial flora of warm-blooded animals and humans (Taucer-Kapteijin et al., 2016). Enterococci can also colonise the genito-urinary tract as well as the oral and vaginal cavities of immune-compromised patients (Manero et al., 2002). These organisms are ubiquitous in nature, thus enterococci can survive in a variety of environmental niches such as soil, water (usually as faecal pollutants), food products and plants (Kühn et al., 1995; Müller et al., 2001; Ateba and Mohapi, 2013; Ateba et al., 2013; Chajęcka-Wierzchowska et al., 2016). In fact, enterococci were extensively screened from cattle and pigs (Manero et al., 2002), dogs, horses and chicken (Hammerum et al., 2000), sheep, swine, rabbits and wild birds (Poeta et al., 2005). Certain species such as the yellow-pigmented E. casseliflavus and E. mundtii are most frequently associated with plants (Müller et al., 2001; Salminen et al., 2004).

In Mafikeng, North West Province, South Africa, enterococci have also been isolated from ground water intended for human consumption and food products (Ateba and Maribeng, 2011; Ateba and Mohapi, 2013; Ateba et al., 2013). Their morphological characteristics can be clearly viewed when cultured on brain heart infusion agar for 18 to 24 hrs at 37°C. They do not produce catalase, except for a few strains (Devriese et al., 2002). In addition, some enterococcal species are motile (E. casseliflavus, E. gallinarum) with a scanty flagellum; enterococci are facultative anaerobes (chemo-organotrophs) and lactic acid producers as a result of glucose fermentation through the Embden-Meyerhof-Parnas pathway (Holt et al., 1994). All species, with the exception of E. faecalis, possess Lysine-D-asparagine bonds; E. faecalis has a peptidoglycan of the lysine-alanine 2-3 type (Domig et al., 2003). Enterococci

(31)

thrive under extreme temperatures, ranging from 5ºC to 50ºC. They display optimal growth at pH 7.5 but can also survive harsh conditions that could be lethal to other bacteria such as media with high salt concentrations. In fact, enterococci grow optimally at 37°C in media supplemented with 6.5% (w/v) NaCl and 40% (w/v) bile salts.

According to Devriese and his colleagues (2002), enterococci can be differentiated through many phenotypic attributes, except for some few strains. For instance, some species have demonstrated intolerance to NaCl (E. avium, E. cecorum and E. columbae). Moreover, the Lancefield group D antigen cannot be verified in numerous isolates that belong to the avium species group and enzymatic activity might vary from one species to the other (Devriese et al., 2002). Bile esculin agar (BEA) has been used for presumptive identification and differentiation of enterococcal isolates from non-group D streptococci based on the fact that enterococci tolerate bile salts and hydrolyse esculin. Esculin iron agar and 0.05% (w/v) K-tellurite agar have been used as alternative selective media for enterococci (Domig et al., 2003). According to Domig and his colleagues (2003), trypticase soy agar and Columbia agar supplemented with 5% (v/v) defibrinated sheep blood can be used to assess enterococcal isolates ability to rupture red blood cells. However, cytolysin mediated hydrolysis demonstrates β-haemolysis on human and horse blood agar (Domig et al., 2003; Mundy et al., 2000). The yellow coloration displayed by some enterococcal species (E. casseliflavus, E. flavescens and E. mundtii) can be assessed using Trypticase/Tryptone soy agar incubated for 18-24hrs at 35°C (Messer and Dufour, 1998). Last but not the least, enterococcal species such as E. faecalis, E. faecium, E. gallinarum and E. casseliflavus produce bacteriocins referred to as enterocins that are active against certain

bacteria (De Vuyst et al., 2003).

E. faecalis is presently the most investigated enterococcal species because of its prevalence in

nosocomial settings (Zhang et al., 2012). Until recently, the only publicly sequenced genome available was that of E. faecalis V583, which was the first reported clinical VRE in the USA

(32)

(Aakra et al., 2005). The genomes of the other enterococcal strains, including E. faecium, E. casseliflavus and E. gallinarum, among others, became available later on (Zhang et al., 2012;

Beukers et al., 2017).

2.2 Characterisation of enterococci

2.2.1 Phenotypic methods of characterisation

A variety of phenotypic methods have been used to characterise enterococci from different sources (Kuzucu et al., 2005). Some of these methods include the assessment of sugar utilization and enzyme production (biotyping) (Tomayko and Murray, 1995), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), multilocus enzyme electrophoresis (MLEE), antimicrobial susceptibility testing, serotyping, long-chain fatty acid analysis, fatty acid methyl esters analysis (FAME), enterocin typing, pyrolysis mass spectrometry (pyMS), vibrational spectroscopic methods and proton magnetic resonance spectroscopy (1H MRS) (Lancefield, 1933; Pompei et al., 1992; Tomayko and Murray, 1995; Goodacre et al., 1996; Morrison et al., 1999; Bourne et al., 2001; Kirschner et al., 2001; Tyrell et al., 2002; Kuzucu et al., 2005; Macovei and Zurek, 2006). These methods are fully discussed

in the following sections.

2.2.1.1 Assessment of sugar utilization and enzyme production (biotyping)

This method of identifying and typing is a traditional way of differentiating bacteria, and enterococci in the present situation since it consists of a battery of bacteriological tubes containing different carbohydrates and indicator dyes and the identity of the isolates relies on a numerical analysis of the results (Manero and Blanch, 1999). A number of miniaturised test kits have been developed to characterise enterococci based on data generated from routine analysis of clinical specimens (Domig et al., 2003). The principle of these kits relies on the ability to produce colour changes from the metabolism of specific types of sugar or when a

(33)

given enzymatic activity occurs (Domig et al., 2003; Salminen et al., 2004). Specific tests kits frequently used include the API 20 Strep (Bio-Merieux, France), the API 50 CH (Bio-Merieux, France) and the Rapid ID32 Strep (Bio-Merieux, France) (Devriese et al., 2002). Moreover, test kits such as the API zym (Bio-Merieux) can be used to determine the potential of isolates to produce the esculinase and pyrase enzymes (Manero et al., 2002). Although these kits are time-saving and some of them such as the PhenePlateTM PhP plate system (PhPlate microplate Techniques, Stockholm, Sweden) have been reported to produce results that are similar to those of pulsed-field gel electrophoresis (PFGE) (Künh et al., 1995), a huge disadvantage is the fact that they merely identify a limited number of Enterococcus species, thus making additional assays necessary for a fine-tuned differentiation.

2.2.1.2 Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Even though phenotypic methods have yielded reliable results in the characterisation of enterococcal strains (Domig et al., 2003). They have proved to be time-consuming and ambiguous (Salminen et al., 2004). Thus, a vast array of methods have been developed to achieve a less time-consuming and a more precise differentiation scheme for these bacterial species (Donabedian et al., 2010). According to Domig and his colleagues (2003), standard SDS-PAGE assays can be use to characterise cellular protein and the generated fingerprints can be used for a rapid screening of strains. This is accurate when comparing and typing isolates at both species and strain levels (Domig et al., 2003; Hendrickx et al., 2009). Relatively cheap and simple, SDS-PAGE has been used to differentiate between strains and has, therefore, become a reference method in the characterisation of enterococci (Latasa et al., 2006).

2.2.1.3 Multilocus enzyme electrophoresis (MLEE)

The Multilocus enzyme electrophoresis (MLEE) is one of the first non-DNA method that has been used for genetic typing of bacterial isolates (Pujol et al., 1997). According to Hall and his colleagues (1996), fifty percent of the enzymes studied until date exist in numerous forms.

(34)

These enzymes may differ slightly in their chemical structures and thus, their kinetic or catalytic properties, providing opportunities for considerable varietions in their electrophoretic mobility patterns (Domig et al., 2003). This is based on the fact that genetic factors determine enzyme multiplicity (Araujo and Sampaio-Maia, 2018) and there is a possibility that isoenzymes could be dispersed among the various cellular parts and could be regulated by no less than two dissimilar genetic factors (Pujol et al., 1997). The presence of manifold loci that regulate enzymes which are specific to a similar substrate, is due to the replication of genetic determinants (Rathnayake et al., 2011) and as a result of these point mutations, duplicated genetic factors cause a discrepancy in the composition of amino acids. The main implication is that different enzyme types can be separated by electrophoresis based on variabilities of their charge or sizes (Domig et al., 2003; Rathnayake et al., 2011).

2.2.1.4 Antimicrobial susceptibility testing

Most enterococci isolated from different sources have demonstrated resistance to antimicrobials (Hughes, 2003). This has urged scientists to assess their antibiotic susceptibility profiles since their disease-causing abilities cannot be underestimated (Huttner et al., 2013). Generally, antibiotic resistance in enterococci and most bacterial species are either intrinsic or acquired from other strains or the environment (Courvalin, 2006). A number of studies have revealed that antibiotic resistance patterns of isolates can be as a tool for characterisation of Enterococcus isolates from diverse sources and geographical locations (Arvanitido, 2003; Choi

et al., 2003). For instance, Choi et al. (2003) and Arvanitido (2003) used the antibiotic

resistance profiles of enterococci to characterise isolates from the Huntington Beach in California, USA and in swimming seawaters in Greece, respectively. The Kirby-Bauer disc diffusion assay is most frequently used to assess antibiotic resistance in isolates (Rathnayake et al., 2011), followed by Frank’s E-test (Franz et al., 2001) and the minimum inhibitory

(35)

2.2.1.5 Serotyping

In 1933, Rebecca Lancefield was the first to report that β-haemolytic faecal streptococci typically possessed the group D antigen (Lancefield, 1933). The recent trend nowadays, is to use the numerical chemotaxonomy and genetic techniques to classify streptococci in taxa (Cocconcelli et al., 1996).

2.2.1.6 Long-chain fatty acid analysis

Long chain fatty acids are a group of important metabolites found in most living cells, which are involved in energy production (Nagy et al., 2004). Gas chromatography or High-pressure liquid chromatography are used to generate long-chain fatty acid profiles of microrganisms (Tyrrell et al., 2002; Nagy et al., 2004). Tyrrell and his colleagues (2002) described the new enterococcal isolates, E. gilvus and E. pallens by comparing their long-chain fatty acid features with that of related group 1 enterococci that also possessed long-chain fatty acid.

2.2.1.7 Fatty acid methyl esters analysis (FAME)

Fatty acid methyl esters analysis (FAME) is the assessment of microbial fatty acid methyl esters extracts using gas chromatography (Lang et al., 2001). Initially, the samples for gas chromatography are prepared through two protocols, which involve extraction and methylation (Lang et al., 2001). FAME profiles generated are used alongside biotyping and ribotyping to analyse enterococcal isolates from cheese (Lang et al., 2001). They were used to demonstrate a significant overlap between the groups, characterised through the aforementioned methods (Chajecka-Wierzchowska et al., 2017).

(36)

Spectroscopic assays come in handy in the characterisation of enterococcal isolates besides the other phenotypic methods. Spectroscopic methods include pyrolysis mass spectrometry (pyMS), vibrational spectroscopic methods and proton magnetic resonance spectroscopy (1H MRS). Spectroscopic methods are used to determine the structure or molecular features of a sample through the interaction of light with matter. The magnetic field around an atom in a molecule is unique and affects its resonance frequency in such a way that the molecular electronic structure of functional compounds such as proteins, can be determined (Kirschner et al., 2001). Morrison and his colleagues (1999) used pyMS to differentiate

Vancomycin-resistant E. faecium strains that have similar PFGE groupings. Moreover, rapid identification of isolates as Enterococcus spp. rather than Streptococcus spp. was achieved by Goodacre et al. (1996) through “Fourier Transform Infrared Spectroscopy” combined with “Artificial Neural Network” (FT-IR, vibrational spectroscopy).

2.2.2 Genotypic methods of characterisation

Despite the fact that phenotypic methods are reasonably efficient in the characterisation of enterococci, genotypic methods have fine-tuned the characterisation of enterococci, mainly due to their high specificity and sensitivity (Kirschner et al., 2001). Genotypic methods are founded on the nucleic acid characteristics of an isolate instead of any phenotypic trait and are highly reproducible and reliable (Rathnayake et al., 2011). Examples of genotypic methods that have been used for the characterisation and typing of enterococci are as follows: “Restriction Endonuclease Analysis” (REA) of the entire chromosome in the DNA (Ke et al., 1999); “Plasmid Profiling” (Hammerum et al., 2000); “Pulsed-field Gel Electrophoresis” (Kirschner et al., 2001); “Ribosomal RNA gene Restriction Analysis” also known as ribotyping (Hughes,

2003); “PCR assays”; Nucleic Acid Hybridisation; Partial Sequence Analysis and Multilocus Sequence Typing (MLST) (Ke et al., 1999; Hammerum et al., 2000; Getachew et al., 2013).

(37)

This technique is among the main methods of characterising genomic DNA that were ever used for genetic typing of bacterial species (Price et al., 2002). Chromosomal DNA from an isolate is extracted and chunked off with a specific restriction enzyme (Price et al., 2002). After analysis, DNA fragments are resolved by agarose gel electrophoresis based on differences on their size, resulting in a banding pattern called DNA fingerprints or DNA profiles (Domig et al., 2003). Eventhough this technique was not recommended as an ideal method, several

authors used REA to type enterococcal isolates. For instance, differentiation of E. faecium strains from different sources has been used to group isolates from different sources into REA-based clusters (Price et al., 2002; Chajecka-Wierzchowska et al., 2017).

2.2.2.2 Plasmid profiling

Eventhough plasmid profiling is not a trustworthy assay for strain characterisation, due to the fact that plasmids are rearranged during conjugation processes (Domig et al., 2003; Mannu et al., 1999; Wardal et al., 2010), to enhance its reliability, plasmid profiling has been used in

combination with Restriction Assay of plasmid DNA and PFGE of genomic DNA, to type E. faecium strains in epidemiological surveys (Domig et al., 2003; Mannu et al., 1999 and

Morrison et al., 1999). It could also be used as a complementary technique to RAPD-PCR (Domig et al., 2003).

2.2.2.3 Pulsed-field gel electrophoresis

This technique is used to separate large DNA fragments obtained through an enzymatic digestion process since they are larger than 20 kb and are thus, able to display the same mobility during an electrophoretic run (Hammerum et al., 2000). Basically, purified genomic DNA is digested with specific restriction enzymes to produce large DNA fragments, ranging from 10 to 800 kb (Gelsomino et al., 2002) that are resolved by electrophoresis under alternating electric fields, with subsequent production of isolate-specific DNA banding patterns (Tomayko et al., 1995). Due to its high discriminatory power, this method is considered a gold standard

(38)

technique for genotyping of bacterial isolates, especially during epidemiological investigations (Tomayko et al., 1995). Although there are many different restriction enzymes available, SmaI and ApaI are the suitable and frequently utilised restriction enzymes that have produced reliable results in the PFGE typing of enterococci (Hammerum et al., 2000). PFGE can be considered as a very effective method for assessing the genetic filiation between various enterococcal strains (Hammerum et al., 2000). Furthermore, it is thought to be an indispensable instrument for the characterisation of enterococci originating from various sources, Vancomycin resistant enterococci from sick individuals and animal food (Gelsomino et al., 2002; Hammerum et al., 2000). PFGE is fairly easy to do, straightforward, reliable and inexpensive but presents, however, some setbacks due to the fact that some bacteria may transform rapidly and get genomic transposons or other genetic material through integration, thus developing dissimilar banding arrangements (Rathnayake et al., 2011).

2.2.2.4 Ribosomal RNA gene Restriction Analysis or Ribotyping

Through this method, nucleic acid probes are used to identify ribosomal genetic elements in bacterial isolates (Price et al., 2002). Ribosomal RNA is inherent to all bacteria and is categorised into three types (23S, 16S and 5S rRNA). The genes encoding for rRNA are highly conserved (Domig et al., 2003) thus, these genetic features are very alike in most bacteria (Price et al., 2002; Oana et al., 2002). The rRNA operons can be present in 2 to 11 copies in a given

bacterium, 5 to 6 operons in the case of enterococci (Domig et al., 2003; Hammerum et al., 2000). Practically, genomic DNA is extracted and digested into smaller sizes with restriction endonucleases; the fragments obtained are separated as they undergo gel electrophoresis (Domig et al., 2003) and dried onto a nylon or nitrocellulose membrane (Domig et al., 2003; Price et al., 2002). Subsequent detection is achived with the aid of probes containing 16S, 23S or 5S rRNA sequences. Each portion of bacterial genetic material containing a gene of the ribosome is highlighted, creating a unic fingerprint. The endonucleases; EcoRI, HindIII, PvuII,

(39)

BamHI and BscI are commonly used in ribotyping analysis (Rathnayake et al., 2011).

Ribotyping is relatively advantageous as compared to other probe-based DNA fingerprinting assays for characterising enterococci due to its potential to allow intraspecies and interspecies discrimination (Price et al., 2002). This is mainly due to the fact that rRNA genes are highly conserved and the use of a particular probe can characterise at subspecies level, all enterococci (Hammerum et al., 2000). However, ribotyping is time-consuming and laborious because many stages are involved and the restriction endonucleases have to be species-specific (Price et al., 2002).

2.2.2.5 PCR-based typing techniques

There are numerous PCR-based typing techniques that have been developed to study genotypic polymorphisms in enterococci and these are outlined in the sections that follow.

2.2.2.5.1 Randomly Amplified Polymorphic (RAPD) DNA - PCR

This typing technique is different from arbitrarily primed PCR (AP-PCR) and DNA amplification fingerprinting (DAF) (Domig et al., 2003). The size of the primers utilised, the quality of the yields obtained and the DNA amplification conditions are specific to each of these techniques (Gelsomino et al., 2002). Arbitrarily designed primers of not more than 7 to 10 base pairs targeting an unspecific genome sequence are used with a subsequent display of polymorphism by the sizes of the amplified genes that were yielded, referred to as “Random Amplified Polymorphic DNA” (RAPD), which could be utilised to compare bacterial strains (Domig et al., 2003). This method differs from classic PCR in the sense that a unic primer is used rather than two and there is a low-stringency annealing temperature. This technique was proved to be very reliable in the screening and characterisation of enterococcal strains from food (Gelsomino et al., 2002 and Mannu et al., 1999) and clinical samples as well as the assessment of the epidemiological factors of VREs.

(40)

2.2.2.5.2 Amplified Fragment Length Polymorphism (AFLP)

This fingerprinting technique is basically a PCR amplification of restriction fragments of digested whole genomic DNA (Vos et al., 1995). First, the whole genomic DNA undergoes restriction and ligation of oligonucleotide adapters; followed by selective amplification and gel assay of the amplified genetic material (Vos et al., 1995). Antonishyn and his colleagues (2002) used a novel fluorescence-based AFLP for the characterisation of Vancomycin-resistant E. faecium strains.

2.2.2.5.3 Rep-PCR

The foundation of this technique is the fact that bacteria harbour particular replicas of DNA sequences dispersed within the genome (Petroziello et al., 1996). These “interspersed repetitive (rep) DNA elements” are separated by various distances and these vary from one bacterium to another. DNA fragments of varying sizes are obtained on subsequent amplification of the regions between the repetitive elements and the PCR products undergo size-separation using gel electrophoresis, yielding DNA specific fingerprint patterns (Lee et al., 1999). This technique was used to produce a DNA fingerprinting pattern of E. faecalis and E. faecium and to type VRE strains by Petroziello et al. (1996) and Lee et al. (1999) respectively.

2.2.2.5.4 PCR-ribotyping

Because spacer regions between 23S, 16S and 5S rRNA genes in genomes of microorganisms are heterogenous, the discriminatory power of this PCR technique is improved when restriction enzymes are used (Rathnayake et al., 2011). Most bacterial classes harbour several replicas of the operon for rRNA in such a way that there is a difference in spacer regions lengths and/or sequences within a particular strain. Thus, Oana et al., (2002) suggested that multiple bands yielded from a single strain after DNA amplification, represent spacer regions of different sizes in different ribosomal RNA coding operons.

(41)

Tyrrell and his colleagues (2000) found that the PCR amplification of the intergenic spacer between 16S and 23S rRNA yield features that characterise enterococci when examined with 6% non-denaturing acrylamide-bisacrylamide gel electrophoresis.

2.2.2.5.6 Amplified ribosomal DNA Restriction Analysis (ARDRA)

This method is founded on the amplification of a gene segment containing the 16S, the spacer region in between 16S and 23S and a portion of the 23S rDNA, with restriction enzyme digestion. This technique was used to type several dairy-related enterococci (Heyndrickx et al., 1996).

2.2.2.5.7 RFLP of PCR-amplified 16S rDNA

This method is a blend of PCR-ribotyping, SDS PAGE and 16S rDNA sequence; it was used to screen enterococci isolated from plants (Müller et al., 2001).

2.2.2.5.8 Broad-range PCR-restriction fragment length polymorphism (PCR-RFLP)

Teng et al. (2001) introduced this method to categorise eight frequently known enterococcal isolates. Restriction fragments were produced by HaeIII and RsaI.

2.2.2.6 PCR-based identification techniques

Genus-specific PCR is used for the characterisation at the genus level by targeting genus genes such as the tuf gene (Ke et al., 1999) while species-specific PCR targets species-specific genes such as the D-alanine/D-alanine ligase (ddl) gene or the groESL gene (Teng et al., 2001 and Domig et al., 2003).

2.2.2.7 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

This technique was developped in order to detect specific mRNA in enterococci, with a particular focus on vanA and vanB genes (Privitera et al., 1999). It is based on the fact that transcription of mRNA associated to vanA gene occurs only once induction with Vancomycin

(42)

or Teicoplanin has occured. Moreover, mRNA associated to vanB gene is only transcribed in the presence of Vancomycin (Domig et al., 2003).

2.2.2.8 Nucleic acid hybridization

rRNA molecules, especially 16S rRNA and 23S rRNA, contain highly conserved regions that are found in all eubacteria as well as regions that are species-specific. This, therefore, gives an outlet to the design from these highly conserved regions, of universal probes or primers specific for all eubacteria. Complementary oligonucleotide probes were synthesised and used by Cocconcelli et al. (1996) to detect strains of E. faecalis and E. faecium in the microflora of cheese. Frahm et al. (2001) suggested a procedure for the screening of enterococci and Pseudomonas aeruginosa in water.

2.2.2.9 Multilocus sequence typing (MLST)

Eventhough the aforementioned typing methods are reliable in the characterisation of enterococci, in some instances, they do present some setbacks (Rathnayake et al., 2011). For instance, comparison of DNA fragments between labs can be difficult and the type of the genetic dissimilarity indexed, is frequently poorly assimilated (Ochoa et al., 2013). MLST solves these issues in the sense that, it relies on the identification of genetic variations (called alleles) in the portions of inner pieces of housekeeping genetic attributes. Each allele is given a number to produce an allelic profile, the allelic profile or grouping of alleles at each loci, determines the sequence type (ST) of each organism and can be used to characterise a specific strain (Dingle et al., 2001; Ochoa et al., 2013). Data are saved, shared and updated from a central database (www.mlst.net). This technique is ideal for the assessment of population structure, mutation and recombination rates within a microbial species. It can also be of great help in the study of host-pathogen relationships (Dingle et al., 2001). However, the disadvantages of this technique lay in the fact that the PCR primers used in this technique, are unique for particular sequences in a species or narrowly related strain groups. Moreover, it is

(43)

expensive because of the expenses related to the DNA polymerase, the sequencing reaction apparatuses and its repairs (Dingle et al., 2001; Rathnayake et al., 2011).

2.2 Pathogenic attributes of enterococci

The sole occurrence of resistance genetic elements in a particular strain is not indicative of its ability to be pathogenic. Rather, the pathogenicity of a strain requires a combination of these genetic attributes with virulence factors (O’Driscoll and Crank, 2015; Heidari et al., 2016). Genes conferring resistance to antimicrobials are harboured on the same mobile genetic elements with genes coding for virulence factors. In fact, virulence factors and antibiotic resistance plasmids are transmitted through very efficient mechanisms of gene transfer (Eaton and Gasson, 2001).

2.2.1 Virulence factors in enterococci

According to Upadhyaya et al., (2009), “pathogenesis of most illnesses does not depend solely on colonisation but requires colonisation, attachment to the host’s cells, invasion of the tissues and resistance to non-specific defensive mechanisms”. There is substantial proof that enterococci that possess virulence factors are more infectious than those without. Two types of virulence factors have been identified and characterised in enterococci as follows: surface factors (involved in the colonisation of the host cell); and substances that cause tissular necrosis (Sava et al., 2010).

2.2.1.1 Virulence factors that promote colonisation of host cells

The adhesion ability of enterococci to their host tissues, coupled with their resistance to low pH and high concentrations of bile salts, makes them one of the most common bacteria in the colon (Tomita and Ike 2004; Foulkié et al., 2006). Their adhesins, without which they could be removed by the peristaltic movement of the intestines, allow their attachment to receptors of the mucosal membranes or to proteins of the extracellular matrix thus, favouring

(44)

colonisation of the epithelial cells (Franz et al., 2003). Some virulence factors that promote colonisation of host cells are as follows: aggregation substance (AS); collagen-binding protein (Ace); cell wall adhesin (EfaA); and enterococcal surface protein (esp) (Strzelecki et al., 2011; Hollenbeck and Rice, 2012).

2.2.1.1.1 Aggregation substance (AS)

Although it is still a current subject of intense research, it is a protein that is encoded on plasmids that act as enterococcal sex pheromones. It facilitates plasmid exchange by mediating aggregation between bacteria (Galli et al., 1990). Sequencing assays have revealed that it encompasses two Arg-Gly-Asp components that play the role of ligands to enterococcal binding substances or “integrins” (Strzelecki et al., 2011). The molecular weight of this peptide is 137 kDa and it displays a hairpin-like structure (Wierzchowska et al., 2017). LPXTG is an important part of its molecular assemblage which is highly conserved and its distinctive sequence is regarded as the site of recognition and cleavage by sortases (Dramsi et al., 2005), which connects them covalently to the cell wall (Dramsi et al., 2005). Aggregation substances or adhesins confer high virulence attributes to enterococci while protecting them from destruction by leukocytes (Rakita et al., 1999; Strzelecki et al., 2011) and thus, are considered as superantigens (Kozlowicz et al., 2006; Clewell et al., 2000; Dunny et al., 1995). Adhesins are involved in the dissemination of plasmids harbouring antimicrobial resistance genetic determinants and other virulence factors such as cytolysin among other enterococci (Wardal et al., 2010). Finally, aggregation substance and cytolysin altogether increase the strain's

virulence by regulating cytolysin through the quorum-sensing system, causing destruction of deeper tissues (Gilmore et al., 2002; Foulquié et al., 2006). The plasmids harbouring genetic determinants that regulate AS proteins are as follows: pAD1 (asa1 protein); pPD1 (asp1 protein); and pCF10 (asc10 protein) (Clewell, 2007; Dunny, 2007).

(45)

With a molecular weight of 74 kDa, Ace (adhesin to collagen) is an adhesin encoded by the ace gene (Rich et al., 1999). This protein could be used to identify species since it was isolated from E. faecalis strains, both from healthy carriers and from people with enterococcal infections (Duh et al., 2001). As it is the case with AS protein, Ace facilitates the adhesion of enterococci during the colonisation process, to proteins of the extracellular matrix; it plays a role mostly in binding type I and IV collagens of (Nallapareddy et al., 2000). Ace is part of the family of surface proteins refered to as “microbial surface component recogniing adhesive matrix molecules” (MSCRAMMs) (Patti et al., 1994; Hendrickx et al., 2009; Nallapareddy et al., 2008).

2.2.1.1.3 Endocarditis specific antigen (EfaA)

Encoded by the efAfs gene in E. faecalis strains and by efArm in E. faecium strains, it is a protein that weighs 34 kDa (Eaton and Gasson, 2001; Sava et al., 2010). This genetic determinant is attached to the afaCBA operon that codes for ABC permease (Abrantes et al., 2013). The EfaA protein shows similarities with the adhesins present in the streptococcal cell wall like the FimA protein of Streptococcus parasanguis, the ScaA in S. gorgonii, the PsaA in S. pneumonia and the SsaB in S. sanguis (Archimbaud et al., 2002). It has been demonstrated

through genetic assays that efaA genes have homologues in E. avium, E. asini, E. durans and E. solitaries strains (Semedo et al., 2003; Jiménez et al., 2013).

2.2.1.1.4 Enterococcal Surface protein (Esp)

Esp is a surface adhesin which weighs about 200 kDa, it is the biggest protein ever to be

screened in enterococci (Toledo-Arena et al., 2001). The esp gene that codes this peptide is present on the pathogenicity island (PAI), which also encompasses proteins involved in the active flushout of antibiotics/antimicrobials (Leavis et al., 2004). This most likely resulted from the horizontal exchange of genes between E. faecalis and E. faecium. The components of Esp protein ressemble that of other adhesins encountered in Gram positive bacteria (Wierzchowska

Assessment of feedlots cattle in the development and spread of Vancomycin resistant Enterococci